Wedekind Joseph E, McKay David B
Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305, USA.
Biochemistry. 2003 Aug 19;42(32):9554-63. doi: 10.1021/bi0300783.
The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.
引导酶是一种通过体外筛选得到的小核酶，它催化位点特异性的、依赖Pb(2+)的RNA切割。活性需要Pb(2+)；Mg(2+)抑制活性，而许多二价和三价离子则增强活性。引导酶结构由一个被三核苷酸凸起打断的RNA双链体组成。在这里，以Mg(2+)作为唯一二价抗衡离子以及以Mg(2+)和Sr(2+)（在结合方面模拟Pb(2+)但不模拟催化作用）确定的分辨率为1.8 Å的晶体结构，揭示了金属离子与引导酶的基态和预催化构象的相互作用。Mg(H₂O)₆²⁺离子通过在大沟中结合一条RNA链的串联嘌呤，在多个位置桥接双链体的互补链。在一个位点，Mg(H₂O)₆²⁺在基态构象中连接三核苷酸凸起的磷酸二酯主链，但在预催化构象中不连接，这表明(a) Mg(2+)可能通过稳定基态来抑制引导酶活性，以及(b) 从该位点取代Mg(2+)的金属离子可能激活引导酶。Sr(2+)与预催化引导酶中假定的催化Pb(2+)位点结合，以一种有助于催化核糖2'-羟基与可切割键对齐以进行切割的方式诱导局部结构变化。这些数据支持一个模型，其中催化离子与引导酶的预催化构象结合，连同三核苷酸凸起的灵活性，可能促进围绕可切割磷酸二酯键的结构重排，有利于允许键切割的构型。
